Our aims are to define the molecular mechanisms for the regulation of cyclic nucleotide metabolism in normal mammalian tissues. Specific emphasis will be placed on the identification, characterization and regulation of several different isozymes of cyclic nucleotide phosphodiesterase, the enzyme(s) that control the breakdown of cyclic nucleotides in the cell. Three major approaches will be emphasized. First, each different isozyme will be isolated, purified, and individually studied in its pure form. Particular attention will be given to those isozymes of phosphodiesterase (PDE) for which no pure proteins have been isolated. These studies will include tests of their control by covalent modification and by allosteric effectors. This should allow information relevant to the individual control of each form in the intact cell to be deduced. A second approach will be to prepare isozyme specific monoclonal antibodies to each of the PDEs and use these antibodies as probes for determining structural and functional properties of the enzymes in crude cell extracts. The antibodies and pure enzymes will also be used to find isozyme specific inhibitors of phosphodiesterase activity. These in turn will be used to assess functional consequences of each isozyme in intact tissues. Finally, questions about the molecular relatedness of each of the isozymes will be addressed by determining primary sequence for several of the isozymes.